Methods for testing milk

ABSTRACT

The disclosure is related generally to methods for testing mammary fluid (including milk) to establish or confirm the identity of the donor of the mammary fluid. Such methods are useful in the milk-bank business to improve safety.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/079,932, filed Apr. 5, 2011, now U.S. Pat. No. 8,278,046, which is acontinuation of U.S. application Ser. No. 12/052,253, filed Mar. 20,2008, now U.S. Pat. No. 7,943,315, which is a continuation-in-part andclaims priority to the international application PCT/US2006/036827 withan international filing date of Sep. 20, 2006, which, in turn, claimspriority under 35 U.S.C. §119 from Provisional Application Ser. Nos.60/719,317, filed Sep. 20, 2005, and 60/731,428, filed Oct. 28, 2005.The disclosures of all of each of which are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The methods featured herein are related generally to methods of testingmammary fluid (including milk) to establish or confirm the identity ofthe donor of the mammary fluid.

BACKGROUND

Unlike blood donors, who give their donation under the directsupervision of the blood bank personnel, human breast milk donors tendto pump their milk for donation at home or other locations convenient tothem and then often store the breast milk in their freezers until theyhave accumulated enough to bring to the donation center. Thus, in theabsence of direct supervision of the donations, questions may arise asto the provenance of the donated breast milk.

In order to establish that the breast milk provided by a donor is, infact, exclusively from that human female donor, some form of testing toestablish donor identity should occur.

Many different methods of DNA typing are known for identifying or typingspecimens from humans. Such methods include short tandem repeats(“STR”), microsatellite repeats or simple sequence repeats (“SSR”)analysis of human DNA; analysis of multiple human genes and analysis ofhuman lymphocyte antigen (HLA) genes and loci by polymerase chainreaction (PCR) analysis, restriction length polymorphism analysis andother methods.

It is known that humans possess antigens which are specific to thatindividual. For example, the human leukocyte antigens (HLA) have beenused in the past for typing tissue for transplantation.

Such typing methods, among others, can be used to test for donoridentity in the methods featured herein.

SUMMARY

The methods and systems featured herein relate to diagnosing orscreening mammary fluid from any number of mammalian organisms. In oneaspect, the invention provides methods and systems for diagnosing orscreening human milk samples to confirm that the milk is from a definedsource.

The methods include obtaining a donated reference sample from apotential mammary fluid donor, e.g., a human breast milk donor. Thesample can be analyzed at or around the time of obtaining the sample forone or more markers that can identify the potential donor.Alternatively, or in addition, the sample can be stored and analyzed foridentifying markers at a later time. When the potential mammary fluiddonor expresses the mammary fluid and donates the fluid (e.g., bybringing or sending the fluid to the donation center), the mammary fluidcan be analyzed for the same marker or markers as the donor's referencesample. The match between the markers (and lack of any additionalunmatched markers) would indicate that the donated milk comes from thesame individual as the one who donated the reference sample. Lack of amatch (or presence of additional unmatched markers) would indicate thatthe donated mammary fluid either comes from a non-tested donor or hasbeen contaminated with fluid from a non-tested donor.

The testing of the reference sample and of the donated mammary fluid canbe carried out at the donation facility and/or milk processing facility.The results of the reference sample tests can be stored and comparedagainst any future donations by the same donor.

Testing donors to confirm their identity improves safety of donatedmilk. It ensures the provenance of the donated milk, which as discussedabove, is most often donated without supervision by the donor center.Testing donor identity by the methods featured herein allows formultiple donations by the same donor, whose identity can be confirmed atthe time of each donation. The donor can live at any distance from thedonation and/or processing facilities, as she can send her milk at longdistances, and her identity can be confirmed based on reference samplesor reference tests stored at the donation and/or processing facility.

The mammary fluid tested by the methods featured herein can be processedfor further use. The donation facility and milk processing facility canbe the same or different facility. The donated milk can be processed,e.g., to obtain human milk fortifiers, standardized human milkformulations; human lipid products, and/or compositions for totalparenteral nutrition.

In one aspect, a method of determining whether a donated mammary fluidwas obtained from a specific subject is featured. The method includes:(a) testing a donated biological sample from the specific subject toobtain at least one reference identity marker profile for at least onemarker; (b) testing a sample of the donated mammary fluid to obtain atleast one identity marker profile for the at least one marker in step(a); and (c) comparing the identity marker profiles, wherein a matchbetween the identity marker profiles indicates that the mammary fluidwas obtained from the specific subject.

Embodiments can include one or more of the following features.

The method can further include: (d) processing the donated mammary fluidwhose identity marker profile has been matched with a reference identitymarker profile. The mammary fluid is human breast milk. Processing caninclude generating a pasteurized milk composition for administration toa human infant. The processing can include: filtering the milk;heat-treating the milk; separating the milk into cream and skim; addinga portion of the cream to the skim; and pasteurizing.

The processed and pasteurized milk composition can include: a humanprotein constituent of about 35-85 mg/mL; a human fat constituent ofabout 60-110 mg/mL; and a human carbohydrate constituent of about 60-140mg/mL, and optionally, one or more constituents selected from the groupconsisting of: calcium, chloride, copper, iron, magnesium, manganese,phosphorus, potassium, sodium, and zinc.

The processed and pasteurized milk composition can include: a humanprotein constituent of about 11-20 mg/mL; a human fat constituent ofabout 35-55 mg/mL; and a human carbohydrate constituent of about 70-120mg/mL, and, optionally, one or more components selected from the groupconsisting of: calcium, chloride, copper, iron, magnesium, manganese,phosphorus, potassium, sodium, and zinc.

The processing can include separating the milk into a cream portion anda skim portion, processing the cream portion, and pasteurizing the creamportion.

The testing of the mammary fluid sample and the testing of thebiological sample can include a nucleic acid typing, e.g., STR analysis,HLA analysis, multiple gene analysis, and a combination thereof.

The donated mammary fluid can be frozen, and the method can includeobtaining the mammary fluid sample by drilling a core through the frozenfluid. Alternatively, or in addition, the method can include obtainingthe mammary fluid sample by scraping the surface of the frozen mammaryfluid.

The method can further include isolating the mammary fluid prior to step(b).

The mammary fluid sample can include a mixture of one or more mammaryfluid samples. The testing of the mammary fluid sample and the testingof the biological sample can include antibody testing to obtain aself-antigen profile. The sample of the donated mammary fluid caninclude a selected solid fraction of the fluid. The identity profilescan include peptide markers. The donated biological sample can be, e.g.,milk, saliva, buccal cell, hair root, and blood.

A lack of a match between the identity marker profiles indicatescontamination of the mammary fluid by another mammal.

Steps (a) through (c) can be carried out at a human breast milk donationcenter or at a milk processing facility.

In another aspect the disclosure is related to a method for determiningwhether breast milk was obtained from a specific human comprisingtesting a sample of the breast milk to obtain an identity marker profileand testing a biological sample from the human to obtain a referenceidentity marker profile and comparing the identity marker profiles.

The disclosure provides a method for determining whether breast milk wasobtained from a desired source or specific human comprising nucleic acidtyping of a sample of the breast milk to obtain a DNA type profile andnucleic acid typing of a biological sample from the human to obtain areference DNA type profile and comparing the DNA type profiles.

In one embodiment, the method of nucleic acid typing of the biologicalsample from the human is selected from STR analysis, HLA analysis ormultiple gene analysis. It is further contemplated that nucleic acidtyping method used for the breast milk sample is the same as that usedfor the biological sample. It is contemplated that the loci/alleles usedfor the DNA type profile will be the same for both the reference DNAtype profile and the breast milk sample DNA type profile.

In one embodiment the breast milk will be frozen. It is furthercontemplated that the method for obtaining the breast milk sample fromthe frozen breast milk will be by drilling a core through the frozenbreast milk. Alternatively, it is contemplated that the breast milksample may be obtained by scraping the surface of the frozen breastmilk.

In another embodiment the breast milk will be liquid. It is contemplatedthat the method for obtaining the breast milk sample will be byisolating the breast milk sample by pipette or other means.

In another embodiment the breast milk samples may be combined or mixedprior to nucleic acid typing.

The disclosure also provides a method for determining whether breastmilk was obtained from a defined source (e.g., a specific human)comprising testing of a sample of the breast milk to obtain aself-antigen profile and testing a biological sample from the human toobtain a reference self-antigen profile and comparing the self-antigenprofiles.

The disclosure also provides an article of manufacture or kit comprisinga container, a label on the container and a reagent for detecting ormeasuring identity markers, wherein the label on the container indicatesthat the reagent can be used to determine the identity marker profile ofbreast milk. In one embodiment, the reagent comprises PCR materials (aset of primers, DNA polymerase and 4 nucleoside triphosphates) thathybridize with the gene or loci thereof. The kit may further compriseadditional components, such as reagents, for detecting or measuring thedetectable entity or providing a control. Other reagents used forhybridization, prehybridization, DNA extraction, visualization and thelike may also be included, if desired. In another embodiment, the regentis an antibody for detecting self-antigens.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of this invention. Indeed, the invention is no way limited tothe methods and materials described herein. For purposes of the methodsfeatured herein, the following terms are defined.

“Mammary fluid” includes breast milk and/or colostrum expressed fromlactating female subjects. Whole mammary fluid, selected liquid or solidfractions of the mammary fluid, whole cells or cellular constituents,proteins, glycoproteins, peptides, nucleotides (including DNA and RNApolynucleotides) and other like biochemical and molecular constituentsof the mammary fluid can be used in the present methods. The mammaryfluid may be obtained from any number of species of female subjectsincluding, but not limited to, humans, bovines, goats and the like.

“Identity marker” includes a marker that can be used to identify anindividual subject from other subjects in a population. Such markers arepresent in the cells found in mammary fluid. Such markers could include,but are not limited to, genes, alleles, loci, antigens polypeptides orpeptides.

An “identity marker profile” comprises a profile of a number of identitymarkers. The profile identifies the individual human or subject fromother humans with a sufficient degree of certainty. It is contemplatedthat the identity marker profile identifies at least one human from100,000 humans, or 1 human from 1 million humans or 1 human from 5million humans.

“Nucleic acid typing” refers to a method of determining the DNA typeprofile of a biological or milk sample. Such methods include, but arenot limited to: STR analysis, HLA analysis or multiple gene analysis ofgenes/alleles/loci present in a polynucleotide sample of the biologicalor milk sample.

“DNA type profile” refers to a profile of a human's or subject's genomicDNA, which is sufficient to distinguish the individual human or subjectfrom other humans with a sufficient degree of certainty. It iscontemplated that the DNA profile identifies at least one human from100,000 humans, or 1 human from 1 million humans or 1 human from 5million humans. Generally the methods featured herein involveidentifying alleles of at least 5 loci/genes or at least 10 loci/genesor at least 13 loci/gene.

An “allele” comprises one of the different nucleic acid sequences of agene at a particular locus on a chromosome. One or more geneticdifferences can constitute an allele. Examples of HLA allele sequencesare set out in Mason and Parham (1998) Tissue Antigens 51:417-66, whichlist HLA-A, HLA-B, and HLA-C alleles and Marsh et al (1992); and Hum.Immunol. 35:1, which list MLA Class II alleles for DRA, DRB, DQA1, DQB1,DPA1, and DPB1.

A “locus” comprises a discrete location on a chromosome. The loci may bepart of a gene or part of repeat sequence. Exemplary human leukocyteantigens (HLAs) loci are the class I MHC genes designated HLA-A, HLA-Band HLA-C; nonclassical class I genes including HLA-E, HLA-F, HLA-G,HLA-H, HLA-J and HLA-X, MIC; and class II genes such as HLA-DP, HLA-DQand HLA-DR. Exemplary STR loci are: CSF1PO, D3S1358, D5S818, D7S820,D8S1179, D13S317, D16S539, DI8S51, D21S11, DYS19, F13A1, FES/FPS, FGA,HPRTB, THO1, TPOX, DYS388, DYS391, DYS392, DYS393, D2S1391, D18S535,D2S.1338, D19S433, D6S477, D1S518, D14S306, D22S684, F13B, CD4, D12S391,D10S220 and D7S523 (see, e.g., U.S. Pat. No. 6,090,558).

A method of HLA analysis or human leukocyte antigen analysis is a methodthat permits the determination or assignment of one or more geneticallydistinct human leukocyte antigen (HLA) genetic polymorphisms by anynumber of methods known in the art. Some methods contemplated aredescribed below.

A method of STR analysis is a method that permits the determination orassignment of one or more genetically distinct STR genetic polymorphismsby any number of methods known in the art. Some methods contemplated aredescribed herein.

A method of multiple gene analysis is a method that permits thedetermination or assignment of one or more genetically distinct geneticpolymorphisms of human genes by any number of methods known in the art.Such genes may or may not include the HLA genes. Some methodscontemplated are described herein.

A number of amplification techniques are known in the art. Amplifyingrefers to a reaction wherein a template nucleic acid, or portionsthereof, is duplicated at least once. Such amplification techniquesinclude arithmetic, logarithmic, or exponential amplification. Theamplification of a nucleic acid can take place using any nucleic acidamplification system, both isothermal and thermal gradient basedincluding, but not limited to, polymerase chain reaction (PCR),reverse-transcription-polymerase chain reaction (RT-PCR), ligase chainreaction (LCR), self-sustained sequence reaction (3SR), andtranscription mediated amplifications (TMA). Typical nucleic acidamplification mixtures (e.g., PCR reaction mixture) include a nucleicacid template that is to be amplified, a nucleic acid polymerase,nucleic acid primer sequence(s), nucleotide triphosphates, and a buffercontaining all of the ion species required for the amplificationreaction.

Amplification products obtained from an amplification reaction typicallycomprise a single stranded or double stranded DNA or RNA or any othernucleic acid products of isothermal and thermal gradient amplificationreactions that include PCR, LCR, and the like.

A “template nucleic acid” refers to a nucleic acid polymer that issought to be copied or amplified. The template nucleic acid(s) can beisolated or purified from a cell, tissue, and the like. The templatenucleic acid can comprise genomic DNA, eDNA, RNA, or the like.

Primers are used in some amplification techniques. A primer comprises anoligonucleotide used in an amplification reaction (e.g., PCR) to amplifya target nucleic acid. The primer is typically single stranded. Theprimer may be from about 5 to 30 nucleic acids in length, more commonlyfrom about 10 to 25 nucleic acids in length.

An STR locus-specific primer is an oligonucleotide that hybridizes to anucleic acid target variant that defines or partially defines thatparticular STR locus.

An HLA allele-specific primer is an oligonucleotide that hybridizes to anucleic acid target variant that defines or partially defines thatparticular HLA allele.

HLA locus-specific primer is an oligonucleotide that permits theamplification of an HLA locus or that can hybridize specifically to anHLA locus.

An allele-specific primer is an oligonucleotide that hybridizes to atarget nucleic acid variant that defines or partially defines thatparticular gene allele.

A locus-specific primer is an oligonucleotide that permits theamplification of a gene locus or that can hybridize specifically to agene locus.

A forward primer and a reverse primer constitute a pair of primers thatcan bind to a template nucleic acid and under proper amplificationconditions produce an amplification product. If the forward primer isbinding to the sense strand then the reverse primer is binding toantisense strand. Alternatively, if the forward primer is binding to theantisense strand then the reverse primer is binding to sense strand. Inessence, the forward or reverse primer can bind to either strand so longas the other reverse or forward primer binds to the opposite strand.

Any number of detectable labels can be used to detect a target nucleicacid by use of amplification or other techniques. A detectable labelrefers to a moiety that is attached through covalent or non-covalenttechniques to an oligonucleotide or other detection agent. Examples ofdetectable labels include a radioactive moiety, a fluorescent moiety, achemiluminescent moiety, a chromogenic moiety and the like. Fluorescentmoieties comprise chemical entities that accepts radiant energy of onewavelength and emits radiant energy at a second wavelength.

Various hybridization techniques can be used in the methods describedherein. Hybridizing or hybridization refers to the binding or duplexingof a molecule to a substantially complementary polynucleotide orfragment through bonding via base pairing. Hybridization typicallyinvolves the formation of hydrogen bonds between nucleotides in onenucleic acid and a complementary second nucleic acid. Methods ofhybridization can include highly stringent, moderately stringent, or lowstringency conditions.

The term “stringent conditions” refers to conditions under which acapture oligonucleotide, oligonucleotide or amplification product willhybridize to its target nucleic acid. “Stringent hybridizationconditions” or “highly stringent conditions” are sequence dependent andwill be different with different environmental parameters (e.g., saltconcentrations, and presence of organics). Generally, stringentconditions are selected to be about 5° C. to 20° C. lower than thethermal melting point (T_(m)) for a specific nucleic acid at a definedionic strength and pH. Stringent conditions are about 5° C. to 10° C.lower than the thermal melting point for a specific nucleic acid boundto a complementary nucleic acid. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a nucleic acid hybridizesto a matched probe. Longer oligonucleotides hybridize at highertemperatures. Typically, stringent conditions will be those in which thesalt concentration comprises about 0.01 to 1.0 M Na (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 300 C for shortprobes (e.g., 10 to 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide. Anextensive guide to the hybridization and washing of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes parts I and II,Elsevier, N.Y.; Choo (ed) (1994) Methods Molecular Biology Volume 33, InSitu Hybridization Protocols, Humana Press Inc., New Jersey; Sambrook etal., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); CurrentProtocols in Molecular Biology, Ausubel et al., eds., (1994).

Hybridization conditions for a particular probe, primer and target arereadily determinable by one of ordinary skill in the art, and generallyis an empirical calculation dependent upon probe length, washingtemperature, and salt concentration. In general, longer probes requirehigher temperatures for proper annealing, while shorter probes needlower temperatures. Hybridization generally depends on the ability ofsingle stranded nucleic acids to anneal with a complementary strandspresent in an environment below their inciting temperature. The higherthe degree of desired homology between a probe and hybridizable target,the higher the relative temperature which can be used. As a result, itfollows that higher relative temperatures would tend to make thereaction conditions more stringent, while lower temperatures less so.

Hybridization wash conditions are ordinarily determined empirically forhybridization of each probe or set of primers to a corresponding targetnucleic acid. The target nucleic acid and probes/primers are firsthybridized (typically under stringent hybridization conditions) and thenwashed with buffers containing successively lower concentrations ofsalts, or higher concentrations of detergents, or at increasingtemperatures until the signal to noise ratio for specific tonon-specific hybridization is high enough to facilitate detection ofspecific hybridization. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C. more usually in excess ofabout 37° C., and occasionally in excess of about 45° C. Stringent saltconditions will ordinarily be less than about 1000 mM, usually less thanabout 500 mM, more usually less than about 400 mM, typically less thanabout 300 mM, typically less than about 200 mM, and more typically lessthan about 150 mM. However, the combination of parameters is moreimportant than the measure of any single parameter. See, e.g., Wetmur etal., J. Mol Biol 31:349-70 (1966), and Wetmur, Critical ReviewsBiochemistry and Molecular Biology 26 (34):227-59 (1991).

In one embodiment, highly stringent conditions comprise hybridization in50% formamide, 6×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (100/μg/ml), 0.5% SDS, and 10% dextransulfate at 420 C, with washes at 42° C. in 2×SSC (sodium chloride/sodiumcitrate) and 0.1% SDS, followed by a high-stringency wash comprising of0.2×SSC containing 0.1% SDS at 42° C.

The terms “complement,” “complementarity” or “complementary,” as usedherein, are used to describe single-stranded polynucleotides related bythe rules of antiparallel base-pairing. For example, the sequence5′-CTAGT-3′ is completely complementary to the sequence 5′-ACTAG-3′.Complementarity may be “partial,” where the base pairing is less than100%, or complementarity may be “complete” or “total,” implying perfect100% antiparallel complementation between two polynucleotides. Byconvention in the art, single-stranded nucleic acid molecules arewritten with their 5′ ends to the left, and their 3′ ends to the right.

The term “complementary base pair” refers to a pair of bases(nucleotides) each in a separate nucleic acid in which each base of thepair is hydrogen bonded to the other. A “classical” (Watson-Crick) basepair contains one purine and one pyrimidine; adenine pairs specificallywith thymine (A-T), guanine with cytosine (G-C), uracil with adenine(U-A). The two bases in a classical base pair are said to becomplementary to each other.

“Substantially complementary” between a probe or primer nucleic acid anda target nucleic acid embraces minor mismatches that can be accommodatedby reducing the stringency of the hybridization media to achieve thedesired degree of hybridization and identification of hybridized targetpolynucleotides.

A “capture oligonucleotide” useful for identification of a targetnucleic acid refers to a nucleic acid or fragment that can hybridize toa polynucleotide, oligonucleotide, amplification product, or the like,and has the ability to be immobilized to a solid phase. A captureoligonucleotide typically hybridizes to at least a portion of anamplification product containing complementary sequences under stringentconditions.

An “HLA locus-specific capture oligonucleotide” is a captureoligonucleotide that is complementary to and hybridizes to a conservedregion of an HLA locus. For example, the capture oligonucleotide can bespecific for the HLA-A locus and will hybridize to one or more conservedregions or subsequences of the HLA-A locus.

Similarly, an “STR locus-specific capture oligonucleotide” is a captureoligonucleotide that is complementary to and hybridizes to a conservedregion of an STR locus. A locus-specific capture oligonucleotide is acapture oligonucleotide that is complementary to and hybridizes to aconserved region of a genetic locus.

A capture oligonucleotide is typically immobilized on a solid phasedirectly or indirectly. Such immobilization may be through covalentand/or non-covalent bonds.

The term “amplicon”, is used herein to mean a population of DNAmolecules that has been produced by amplification, e.g., by PCR.

The term “subject” refers to a lactating mammalian subject. The subjectmay be a human, bovine, goat and the like. For example, the screening asto the origination of milk products from non-human mammals may beimportant for the tracing of products to a particular bovine, forexample, for FDA or other purposes.

“Mutation” as used herein sometimes refers to a functional polymorphismthat occurs in the population, and is strongly correlated to a gene.“Mutation” is also used herein to refer to a specific site and type offunctional polymorphism, without reference to the degree of risk thatparticular mutation poses to an individual for a particular disease.

A “self-antigen” is an antigen which identifies the individual subjectfrom other subjects in the population. Exemplary self-antigens includethe major histocompatibility antigens (MHC) antigens or the blood typeantigens (ABO).

A “self antigen profile” means a profile of a human's or subject'sself-antigens which is sufficient to distinguish the individual subjectfrom other subjects with a sufficient degree of certainty.

The term “antibody” is used in its broadest sense and covers polyclonalantibodies, monoclonal antibodies, single chain antibodies and antibodyfragments.

The details of one or more embodiments of the methods featured hereinare set forth in the description below. Other features, objects, andadvantages of the methods will be apparent from the description and theclaims.

All patents, patent applications, and references cited herein areincorporated in their entireties by reference.

DETAILED DESCRIPTION

In one aspect, the methods featured herein are used to determine milkorigination. For example, in order to ensure that human breast milkreceived from a specific human actually comes from that human, methodsof identity testing are needed on samples of milk received from eachhuman. Testing donors to confirm their identity improves safety ofdonated milk. It ensures the provenance of the donated milk, which asdiscussed above, is most often donated without supervision of personnelof the organization that will be receiving the milk, e.g., a milk bankcenter. Testing donor identity by the methods featured herein allows formultiple donations by the same donor, whose identity can be confirmed atthe time of each donation. The donor can live at any distance from thedonation and/or processing facilities, as she can send her milk at longdistances, and her identity can be confirmed based on reference samplesor reference test results stored at the donation and/or processingfacility.

As part of the qualification process for donating milk, each potentialmilk donor will be identified by biological methods (e.g., biologicalfingerprinting, as described herein). The identifying characteristics ofthe individual (i.e., at least one identity marker) will also be presentin the donor's milk. Such characteristics will be used to match thedonated milk with a specific donor.

Obtaining a Reference Biological Sample

The methods featured herein include, inter alia, obtaining at least onedonated reference sample from a potential mammary fluid donor, e.g., ahuman breast milk donor. Such sample may be obtained by methods known inthe art such as, but not limited to, a cheek swab sample of cells, or adrawn blood sample, milk, saliva, hair roots, or other convenienttissue. Samples of reference donor nucleic acids (e.g., genomic DNA) canbe isolated from any convenient biological sample including, but notlimited to, milk, saliva, buccal cells, hair roots, blood, and any othersuitable cell or tissue sample with intact interphase nuclei ormetaphase cells. The sample is labeled with a unique reference number.The sample can be analyzed at or around the time of obtaining the samplefor one or more markers that can identify the potential donor. Resultsof the analysis can be stored, e.g., on a computer-readable medium.Alternatively, or in addition, the sample can be stored and analyzed foridentifying markers at a later time.

It is contemplated that the biological reference sample may be DNA typedby methods known in the art such as STR analysis of STR loci, HLAanalysis of HLA loci or multiple gene analysis of individualgenes/alleles (further discussed below). The DNA-type profile of thereference sample is recorded and stored, e.g., on a computer-readablemedium.

It is further contemplated that the biological reference sample may betested for self-antigens using antibodies known in the art or othermethods to determine a self-antigen profile. The antigen (or anotherpeptide) profile can be recorded and stored, e.g., on acomputer-readable medium.

Testing a Sample of Donated Mammary Fluid

A subject desiring to donate mammary fluid will express the mammaryfluid (breast milk) using standard procedures. The mammary fluid istypically collected in containers useful for shipping and storage. Themammary fluid can be frozen prior to donation. The mammary fluid may befrozen at the donation facility or processing facility for lateranalysis and use or analyzed without freezing. One or more of thecontainers with donated fluid can be used for obtaining a test sample.The test sample is taken for identification of one or more identitymarkers.

Methods of obtaining a sample of expressed frozen fluid include astainless steel boring tool used to drill a core the entire length ofthe container. Alternatively, a sample may be scraped from the surfaceof the frozen mammary fluid. The container may contain a separateportion for collection of a sample of the expressed mammary fluid, andthis portion may be removed as the sample for testing. Where the mammaryfluid is in liquid form it is contemplated that the method for obtainingthe test sample will be by pipette or other means.

A sample of the donated the mammary fluid is analyzed for the samemarker or markers as the donor's reference sample. The marker profilesof the reference biological sample and of the donated mammary fluid arecompared. The match between the markers (and lack of any additionalunmatched markers) would indicate that the donated milk comes from thesame individual as the one who donated the reference sample. Lack of amatch (or presence of additional unmatched markers) would indicate thatthe donated mammary fluid either comes from a non-tested donor or hasbeen contaminated with fluid from a non-tested donor.

The donated mammary fluid sample and the donated reference biologicalsample can be tested for more than one marker. For example, each samplecan be tested for multiple DNA markers and/or peptide markers. Bothsamples, however, need to be tested for at least some of the samemarkers in order to compare the markers from each sample.

Thus, the reference sample and the donated mammary fluid sample may betested for the presence of differing identity marker profiles. If thereare no identity marker profiles other than the identity marker profilefrom the expected subject, it generally indicates that there was nofluid (e.g., milk) from other humans or animals contaminating thedonated mammary fluid. If there are signals other than the expectedsignal for that subject, the results are indicative of contamination.Such contamination will result in the mammary fluid (e.g., milk) failingthe testing.

The testing of the reference sample and of the donated mammary fluid canbe carried out at the donation facility and/or milk processing facility.The results of the reference sample tests can be stored and comparedagainst any future donations by the same donor.

It is contemplated that samples from a number of milk containers fromthe same subject may be pooled for identity marker testing. It iscontemplated that at least 2 samples, at least 5 samples or at least 8samples may be pooled for testing.

It is contemplated that the test sample of the donated mammary fluid maybe tested by nucleic acid typing using methods known in the art, suchas, STR analysis of STR loci, HLA analysis of HLA loci or multiple geneanalysis of individual genes/alleles to obtain the DNA-type of the milksample. The donated mammary fluid can also be tested for peptideprofiles, e.g., antigen profile.

The DNA-type or another biological profile (i.e., identity profile (s))of the donated mammary fluid test sample (s) will be compared to thereference DNA-type or another biological profile for the putative donor.A match or identity of the DNA-type or biological profile will indicatethat the mammary fluid was obtained from a same (i.e., a specifiedsubject).

Use of the Donated and Tested Mammary Fluid

The mammary fluid tested by the methods featured herein can be processedfor further use. The donation facility and milk processing facility canbe the same or different facility. The donated milk whose provenance hasbeen confirmed can be processed, e.g., to obtain human milk fortifiers,standardized human milk formulations, and/or human lipid compositions.As discussed above, testing the mammary fluid to confirm the identity ofthe donor ensures safety of the mammary fluid and any products derivedfrom such fluid.

Processing of human milk to obtain human milk fortifiers (e.g.,PROLACTPLUS™ Human Milk Fortifiers, e.g., PROLACT+4™, PROLACT+6™,PROLACT+8™, and/or PROLACT+10™, which are produced from human milk andcontain various concentrations of nutritional components) and thecompositions of the fortifiers are described in U.S. patent applicationSer. No. 11/947,580, filed on Nov. 29, 2007, the contents of which areincorporated herein in their entirety. These fortifiers can be added tothe milk of a nursing mother to provide an optimal nutritional contentof the milk for, e.g., a preterm infant. Depending on the content ofmother's own milk, various concentrations of the fortifiers can be addedto mother's milk.

Methods of obtaining standardized human milk formulations (exemplifiedby PROLACT20™, NEO20™, and/or PROLACT24) and formulations themselves arealso discussed in U.S. patent application Ser. No. 11/947,580, filed onNov. 29, 2007, the contents of which are incorporated herein in theirentirety. These standardized human milk formulations can be used tofeed, e.g., preterm infants, without mixing them with other fortifiersor mills. They provide a nutritional human-derived formulation and cansubstitute for mother's milk.

Compositions that include lipids from human milk, methods of obtainingsuch compositions, and methods of using such compositions to providenutrition to patients are described in PCT Application PCT/US07/86973filed on Dec. 10, 2007, the contents of which are incorporated herein intheir entirety.

Methods of obtaining other nutritional compositions from human milk thatcan be used with the methods featured herein are discussed in U.S.patent application Ser. No. 11/012,611, filed on Dec. 14, 2004, andpublished as U.S. 2005/0100634 on May 12, 2005, the contents of whichare incorporated herein in their entirety.

Processing of milk that has been tested with the methods featured hereincan be carried out with large volumes of donor milk, e.g., about 75liters/lot to about 2,000 liters/lot of starting material.

The methods featured herein can also be integrated with methods offacilitating collection and distribution of human milk over a computernetwork, e.g., as described in U.S. patent application Ser. No.11/526,127, filed on Sep. 22, 2006, and published as U.S. 2007/0098863on May 3, 2007; and in U.S. patent application Ser. No. 11/679,546,filed on Feb. 27, 2007, and published as U.S. 2007/0203802 on Aug. 30,2007. The contents of both applications are incorporated herein in theirentireties.

Nucleic Acid Identity Marker Profiles

As discussed above, samples of reference donor nucleic acids (e.g.,genomic DNA) are isolated from any convenient biological sampleincluding, but not limited to, milk, saliva, buccal cells, hair roots,blood, and any other suitable cell or tissue sample with intactinterphase nuclei or metaphase cells.

Methods for isolation of nucleic acids (e.g., genomic DNA) from thesevarious sources are described in, for example, Kirby, DNAFingerprinting, An Introduction, W. H. Freeman & Co. New York (1992).Nucleic acids (e.g., genomic DNA) can also be isolated from culturedprimary or secondary cell cultures or from transformed cell linesderived from any of the aforementioned tissue samples.

Samples of RNA can also be used. RNA can be isolated as described inSambrook et al., supra, RNA can be total cellular RNA, mRNA, poly A+RNA, or any combination thereof. For best results, the RNA is purified,but can also be unpurified cytoplasmic RNA. RNA can be reversetranscribed to form DNA which is then used as the amplificationtemplate, such that PCR indirectly amplifies a specific population ofRNA transcripts. See, e.g., Sambrook et al., supra, and Berg et al.,Hum. Genet. 85:655-658 (1990).

Short tandem repeat (STR) DNA markers, also referred to asmicrosatellites or simple sequence repeats (SSRs) or DNA tandemnucleotide repeat (“DTNR”), comprise tandem repeated DNA sequences witha core repeat of 2-6 base pairs (bp). STR markers are readily amplifiedduring PCR by using primers that bind in conserved regions of the genomeflanking the repeat region.

Commonly sized repeats include dinucleotides, trinucleotides,tetranucleotides and larger. The number of repeats occurring at aparticular genetic locus varies from a few to hundreds depending on thelocus and the individual. The sequence and base composition of repeatscan vary significantly, including a lack of consistency within aparticular nucleotide repeat locus. Thousands of STR loci have beenidentified in the human genome and have been predicted to occur asfrequently as once every 15 kb. Population studies have been undertakenon dozens of these STR markers as well as extensive validation studiesin forensic laboratories. Specific primer sequences located in theregions flanking the DNA tandem repeat region have been used to amplifyalleles from STR loci via the polymerase chain reaction (“PCR”). The PCRproducts include the polymorphic repeat regions, which vary in lengthdepending on the number of repeats or partial repeats, and the flankingregions, which are typically of constant length and sequence betweensamples.

The number of repeats present for a particular individual at aparticular locus is described as the allele value for the locus. Becausemost chromosomes are present in pairs, PCR amplifications of a singlelocus commonly yields two different sized PCR products representing twodifferent repeat numbers or allele values. The range of possible repeatnumbers for a given locus, determined through experimental sampling ofthe population, is defined as the allele range, and may vary for eachlocus, e.g., 7 to 15 alleles. The allele PCR product size range (allelesize range) for a given locus is defined by the placement of the two PCRprimers relative to the repeat region and the allele range. Thesequences in regions flanking each locus must be fairly conserved inorder for the primers to anneal effectively and initiate PCRamplification. For purposes of genetic analysis di-, tri-, andtetranucleotide repeats in the range of 5 to 50 are typically utilizedin screens. Forensic laboratories use tetranucleotide loci (i.e., 4 bpin the repeat) due to the lower amount of “stutter” produced during PCR(Stutter products are additional peaks that can complicate theinterpretation of DNA mixtures by appearing in front of regular allelepeaks). The number of repeats can vary from 3 or 4 repeats to more than50 repeats with extremely polymorphic markers. The number of repeats andhence the size of the PCR product, may vary among samples in apopulation making STR markers useful in identity testing of geneticmapping studies.

There are 13 core STR loci identified in the United States CODISdatabase. These STR loci are THO1, TPOX, CSF1PO, VWA, FGA, D3S1358,D5S818, D7S820, D13S317, D16S539, D8S1179, D18S51 and D21S11. Thesex-typing marker amelogenin, is also included in the STR multiplexesthat cover the 13 core STR loci. The 13 CODIS STR loci are covered bythe Profiler Plus™ and COfiler™ kits from Applied Biosystems (ABI)(Foster City, Calif.). It is contemplated that the following STR locimay be used in this invention: CSF1PO, D3S1358, D5S818, D7S820, D8S1179,D13S317, D16S539, D18S51, D21S11, DYS19, F13A1, FESfFPS, FGA, HPRTB,THO1, TPOX, DYS388, DYS391, DYS392, DYS393, D2S1391, D18S535, D2S1338,D19S433, D6S477, D1S518, D14S306, D22S684, F13B, CD4, D12S391, D10S220and D7S523 (the sequence of each loci is incorporated herein byreference). With the exception of D3 S1358, sequences for the STR lociof this invention are accessible to the general public through GenBank(see U.S. Pat. No. 6,090,558, incorporated herein by reference). OtherSTR loci have been developed by commercial manufacturers and studiedextensively by forensic scientists. These include all of the GenePrint™tretranucleotide STR systems from Promega Corporation (Madison Wis.).

Many different primers have been designed for various STR loci andreported in the literature. These primers anneal to DNA segments outsidethe DNA tandem repeat region to produce PCR products containing thetandem repeat region. These primers were designed with polyacrylamidegel electrophoretic separation in mind as a method ofdetection/measurement, because DNA separations have traditionally beenperformed by slab gel or capillary electrophoresis. STR multiplexanalysis is usually performed with PCR amplification and detection ofmultiple markers. STR multiplexing is most commonly performed usingspectrally distinguishable fluorescent tags and/or non-overlapping PCRproduct sizes. Multiplex STR amplification in one or two PCR reactionswith fluorescently labeled primers and measurement with gel or capillaryelectrophoresis separation and laser induced fluorescence detection is astandard method. The STR alleles from these multiplexed PCR productstypically range in size from 100-800 bp with commercially availablelots.

Gel-based systems are capable of multiplexing the analysis of 2 or moreSTR loci using two approaches. The first approach is to size partitionthe different PCR product loci. Size partitioning involves designing thePCR primers used to amplify different loci so that the allele PCRproduct size range for each locus covers a different and separable partof the gel size spectrum. As an example, the PCR primers for Locus Amight be designed so that the allele size range is from 250 to 300nucleotides, while the primers for Locus B are designed to produce anallele size range from 340 to 410 nucleotides.

The second approach to multiplexing 2 or more STR loci on gel-basedsystems is the use of spectroscopic partitioning. Current state of theart for gel-based systems involves the use of fluorescent dyes asspecific spectroscopic markers for different PCR amplified loci.Different chromophores that emit light at different color wavelengthsprovide a method for differential detection of two different PCRproducts even if they are exactly the same size, thus 2 or more loci canproduce PCR products with allele size ranges that overlap. For example,Locus A with a green fluorescent tag produces an allele size range from250 to 300 nucleotides, while Locus B with a red fluorescent tagproduces an allele size range of 270 to 330 nucleotides. A scanning,laser-excited fluorescence detection device monitors the wavelength ofemissions and assigns different PCR product sizes, and theircorresponding allele values, to their specific loci based on theirfluorescent color.

It is contemplated that a mass spectrometry approach to STR typing andanalysis, examining smaller nucleic acid oligomers may be used becausethe sensitivity of detection and mass resolution are superior withsmaller oligomers. Application of STR analysis to time of flight-massspectrometry (TOF-MS) requires the development of primer sets thatproduce small PCR products 50 to 160 nucleotides in length, typicallyabout 50 to 100 nucleotides in length. Amplified nucleic acids may alsobe used to generate single stranded products that are in the desiredsize range for TOF-MS analysis by extending a primer in the presence ofa chain termination reagent. A typical class of chain terminationreagent commonly used by those of skill in the art is thedideoxynucleotide triphosphates. Again, application of STR analysis toTOF-MS requires that the primer be extended to generate products of 50to 160 nucleotides in size, and typically about 50 to 100 nucleotides inlength (see U.S. Pat. No. 6,090,558 incorporated by reference).

A biotinylated cleavable oligonucleotide is used as a primer in eachassay and is incorporated through standard nucleic acid amplification(i.e., PCR) methodologies into the final product which is measured inthe mass spectrometer. This process is described in, for example, U.S.Pat. No. 5,700,642 and U.S. Pat. No. 6,090,558 (see also Butler et al.,International Journal of Legal Medicine 112(1) 45-59 (1998)). The STRassay involves a PCR amplification step where one of the primers isreplaced by a cleavable biotinylated primer. The biotinylated PCRproduct is then captured on streptavidin-coated magnetic beads forpost-PCR sample cleanup and salt removal, followed by mass spectrometryanalysis.

Single nucleotide polymorphisms (SNPs) represent another form of DNAvariation that is useful for human identity testing. SNPs are the mostfrequent form of DNA sequence variation in the genomes of organisms andare becoming increasingly popular genetic markers for genome mappingstudies and medical diagnostics. SNPs are typically bi-allelic with twopossible nucleotides (nt) or alleles at a particular site in the genome.Because SNPs are less polymorphic (i.e., have fewer alleles) than thecurrently used STR markers, more SNP markers are required to obtain thesame level of discrimination between samples. Approximately 30-50unlinked SNPs may be required to obtain the matching probabilities of 1in 100 billion as seen with the 13 CODIS STRs.

A SNP assay typically involves a three-step process: (1) PCRamplification (2) phosphatase removal of nucleotides, and (3) primerextension using a biotinylated cleavable primer with dideoxynucleotidesfor single-base addition of the nucleotide (s) complementary to theone(s) at the SNP site (Li et al., Electrophoresis 20(6): 1258-1265(1999)).

Simultaneous analysis of multiple SNP markers (i.e. multiplexing) ispossible by simply putting the cleavage sites at different positions inthe various primers so that they do not overlap on a mass scale.

The most common means for amplification is polymerase chain reaction(PCR), as described in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,965,188each of which is hereby incorporated by reference in its entirety. IfPCR is used to amplify the target regions in blood cells, heparinizedwhole blood should be drawn in a sealed vacuum tube kept separated fromother samples and handled with clean gloves. For best results, bloodshould be processed immediately after collection; if this is impossible,it should be kept in a sealed container at about 4° C. until use. Cellsin other physiological fluids may also be assayed. When using any ofthese fluids, the cells in the fluid should be separated from the fluidcomponent by centrifugation.

To amplify a target nucleic acid sequence in a sample by PCR, thesequence must be accessible to the components of the amplificationsystem. One method of isolating target DNA is crude extraction which isuseful for relatively large samples. Briefly, mononuclear cells fromsamples of blood, buccal cells, or the like are isolated. The pelletsare stored frozen at −200C until used (U.S. Patent Publication No.20040253594 which is incorporated by reference).

The pellets may be resuspended in lysis solution from the PUREGENE® DNAisolation kit (Cat#D-5000, GENTRA, Minneapolis, Minn.) containing100/ug/ml of proteinase K. After incubating at 55° C. overnight, DNAextraction is performed according to manufacturers recommendations. TheDNA samples are resuspended in aqueous solution and stored at −200C.

When the sample contains a large number of cells, extraction may beaccomplished by methods as described in Higuchi, “Simple and RapidPreparation of Samples for PCR”, in PCR Technology, p. 31-43 Ehrlich, H.A. (ed.), Stockton Press, New York.

A relatively easy procedure for extracting DNA for PCR is a salting outprocedure adapted from the method described by Miller et al., NucleicAcids Res. 16:1215 (1988), which is incorporated herein by reference.Nucleated cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl,400 mM NaCl, 2 mM Na2 EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution ofproteinase K and 200 ul of a 20% SDS solution are added to the cells andthen incubated at 37° C. overnight. Following adequate digestion, one mlof a 6M NaCl solution is added to the sample and vigorously mixed. Theresulting solution is centrifuged for 15 minutes at 3000 rpm. The pelletcontains the precipitated cellular proteins, while the supernatantcontains the DNA. The supernatant is removed to a 15 ml tube thatcontains 4 ml of isopropanol. The contents of the tube are mixed gentlyuntil the water and the alcohol phases have mixed and a white DNAprecipitate has formed. The DNA precipitate is placed in distilled waterand dissolved. (U.S. Patent Publication No. 20040253594 which isincorporated by reference).

Kits for the extraction of high-molecular weight DNA for PCR includePUREGENE® DNA Isolation kit (D-5000) GENTRA, a Genomic Isolation Kit A.S.A. P.® (Boehringer Mannheim, Indianapolis, Ind.), Genomic DNAIsolation System (GIBCO BRL, Gaithdrsburg, Md.), ELU-QUIK® DNAPurification Kit (Schleicher & Schnell, Keene, N.H.), DNA Extraction Kit(Stratagene, LaJolla, Calif.), TURBOGEN® Isolation Kit (Invitrogen, SanDiego, Calif.), and the like. Use of these kits according to themanufacturer's instructions is generally acceptable for purification ofDNA prior to practicing the methods of the invention (U.S. PatentPublication No. 20040253594 which is incorporated by reference).

The concentration and purity of the extracted DNA can be determined byspectrophotometric analysis of the absorbance of a diluted aliquot at260 nm and 280 nm.

After extraction of the DNA, PCR amplification may proceed. The firststep of each cycle of the PCR involves the separation of the nucleicacid duplex formed by the primer extension. Once the strands areseparated, the next step in PCR involves hybridizing the separatedstrands with primers that flank the target sequence. The primers arethen extended to form complementary copies of the target strands. Forsuccessful PCR amplification, the primers are designed so that theposition at which each primer hybridizes along a duplex sequence is suchthat an extension product synthesized from one primer, when separatedfrom the template (complement), serves as a template for the extensionof the other primer. The cycle of denaturation, hybridization, andextension is repeated as many times as necessary to obtain the desiredamount of amplified nucleic acid (U.S. Patent Publication No.20040253594 which is incorporated by reference).

In one embodiment of PCR amplification, strand separation is achieved byheating the reaction to a sufficiently high temperature for a sufficienttime to cause the denaturation of the duplex but not to cause anirreversible denaturation of the polymerase (see U.S. Pat. No.4,965,188, incorporated herein by reference). Typical heat denaturationinvolves temperatures ranging from about 80° C. to about 105° C. fortimes ranging from seconds to minutes. Strand separation, however, canbe accomplished by any suitable denaturing method including physical,chemical, or enzymatic means. Strand separation may be induced by ahelicase, for example, or an enzyme capable of exhibiting helicaseactivity. For example, the enzyme RecA has helicase activity in thepresence of ATP. The reaction conditions suitable for strand separationby helicases are known in the” art (see Kuhn et al., 1979,CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev,Genetics 16:405-437, incorporated by reference).

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of fourdeoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP)in a reaction medium comprised of the appropriate salts, metal cations,and pH buffering systems. Suitable polymerizing agents are enzymes knownto catalyze template-dependent DNA synthesis. In some cases, the targetregions may encode at least a portion of a protein expressed by thecell. In this instance, mRNA may be used for amplification of the targetregion. Alternatively, PCR can be used to generate a cDNA library fromRNA for further amplification, the initial template for primer extensionis RNA. Polymerizing agents suitable for synthesizing a complementarycopy-DNA (cDNA) sequence from the RNA template are reverse transcriptase(RT), such as avian myeloblastosis virus RT, Moloney murine leukemiavirus RT, or Thermus thermophilus (Tth) DNA polymerase, a thermostableDNA polymerase with reverse transcriptase activity marketed by PerkinElmer Cetus, Inc. Typically, the genomic RNA template is heat degradedduring the first denaturation step after the initial reversetranscription step leaving only DNA template. Suitable polymerases foruse with a DNA template include, for example, E. coli DNA polymerase Ior its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taqpolymerase, a heat-stable DNA polymerase isolated from Thermus aquaticusand commercially available from Perkin Elmer Cetus, Inc. The latterenzyme is widely used in the amplification and sequencing of nucleicacids. The reaction conditions for using Taq polymerase are known in theart (U.S. Patent Publication No. 20040253594 which is incorporated byreference).

Allele-specific PCR differentiates between target regions differing inthe presence or absence of a variation or polymorphism. PCRamplification primers are chosen which bind only to certain alleles ofthe target sequence. This method is described by Gibbs, Nucleic AcidRes. 17:2437-2448 (1989) (U.S. Patent Publication No. 20040253594 whichis incorporated by reference).

Further diagnostic screening methods employ the allele-specificoligonucleotide (ASO) screening methods, as described by Saiki et al.,Nature 324:163-166 (1986). Oligonucleotides with one or more base pairmismatches are generated for any particular allele. ASO screeningmethods detect mismatches between variant target genomic or PCRamplified DNA and non-mutant oligonucleotides, showing decreased bindingof the oligonucleotide relative to a mutant oligonucleotide.Oligonucleotide probes can be designed that under low stringency willbind to both polymorphic forms of the allele, but which at highstringency, bind to the allele to which they correspond. Alternatively,stringency conditions can be devised in which an essentially binaryresponse is obtained, i.e., an ASO corresponding to a variant form ofthe target gene will hybridize to that allele, and not to the wildtypeallele (U.S. Patent Publication No. 20040253594 which is incorporated byreference).

Target regions of a subject's DNA can be compared with the mammary fluidsample by ligase-mediated allele detection. Ligase may also be used todetect point mutations in the ligation amplification reaction describedin Wu and Wallace., Genomics 4:560-569 (1989). The ligationamplification reaction (LAR) utilizes amplification of specific DNAsequence using sequential rounds of template dependent ligation asdescribed in Barany, Proc. Nat. Acad. Sci. 88:189-193 (1990) and U.S.Patent Publication No. 20040253594 which are incorporated by reference.

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. DNA molecules melt in segments, termed melting domains,under conditions of increased temperature or denaturation. Each meltingdomain melts cooperatively at a distinct, base-specific meltingtemperature (TM). Melting domains are at least 20 base pairs in length,and may be up to several hundred base pairs in length.

Differentiation between alleles based on sequence specific meltingdomain differences can be assessed using polyacrylamide gelelectrophoresis, as described in Myers et al., Chapter 7 of Erlich, ed.,PCR Technology, W.H. Freeman and Co., New York (1989) incorporated byreference.

Generally, a target region to be analyzed by denaturing gradient gelelectrophoresis is amplified using PCR primers flanking the targetregion. The amplified PCR product is applied to a polyacrylamide gelwith a linear denaturing gradient as described in Myers et al., Meth.Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, APractical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139(1988). The electrophoresis system is maintained at a temperatureslightly below the Tm of the melting domains of the target sequences.

In an alternative method of denaturing gradient gel electrophoresis, thetarget sequences may be initially attached to a stretch of GCnucleotides, termed a GC clamp, as described by Myers in Chapter 7 ofErlich, PCT Technology Stockton Press, It is contemplated that at least80% of the nucleotides in the GC clamp are either guanine or cytosine.The GC clamp may be at least 30 bases long. This method is particularlysuited to target sequences with high Tm's.

Generally, the target region is amplified by the polymerase chainreaction as described above. One of the oligonucleotide PCR primerscarries at its 5′ end, the GC clamp region, at least 30 bases of the GCrich sequence, which is incorporated into the 5′ end of the targetregion during amplification. The resulting amplified target region isrun on an electrophoresis gel under denaturing gradient conditions asdescribed above. Nucleic acid fragments differing by a single basechange will migrate through the gel to different positions, which may bevisualized by ethidium bromide staining.

Temperature gradient gel electrophoresis (TGGE) is based on the sameunderlying principles as denaturing gradient gel electrophoresis, exceptthe denaturing gradient is produced by differences in temperatureinstead of differences in the concentration of a chemical denaturant.Standard TGGE utilizes an electrophoresis apparatus with a temperaturegradient running along the electrophoresis path. As samples migratethrough a gel with a uniform concentration of a chemical denaturant,they encounter increasing temperatures. An alternative method of TGGE,temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses asteadily increasing temperature of the entire electrophoresis gel toachieve the same result. As the samples migrate through the gel thetemperature of the entire gel increases, leading the samples toencounter increasing temperature as they migrate through the gel.Preparation of samples, including PCR amplification with incorporationof a GC clamp, and visualization of products are the same as fordenaturing gradient gel electrophoresis (see, e.g., U.S. PatentApplication No. 20040253594).

The human leukocyte antigen complex (also known as the majorhistocompatibility complex) spans approximately 3.5 million base pairson the short arm of chromosome 6. It is divisible into 3 separateregions which contain the class I, the class II and the class III genes.In humans, the class I HLA complex is about 2000 kb long and containsabout 20 genes. Within the class I region exist genes encoding the wellcharacterized class I MHC molecules designated HLA-A, HLA-B and HLA-C.In addition, there are nonclassical class I genes that include HLA-E,HLA-F, HLA-G, HLA-H, HLA-J and JLA-X as well as a new family known asMIC. The class II region contains three genes known as the HLA-DP,HLA-DQ and HLA-DR loci. These genes encode the chains of the classicalclass II MHC molecules designated HLA-DR, DP and DQ. In humans,nonclassical genes designated DM, DN and DO have also been identifiedwithin class II. The class III region contains a heterogeneouscollection of more than 36 genes. Several complete components areencoded by three genes including the TNFs (see, e.g., U.S. Pat. No.6,670,124 incorporated by reference).

Any given copy of human chromosome 6 can contain many differentalternative versions of each of the preceding genes and thus can yieldproteins with distinctly different sequences. The loci constituting theMHC are highly polymorphic, that is, many forms of the gene or allelesexist at each locus. Several hundred different allelic variants of classI and class II MHC molecules have been identified in humans. However,any one individual only expresses up to 6 different class I moleculesand up to 12 different class II molecules.

The foregoing regions play a major role in determining whethertransplanted tissue will be accepted as self (histocompatible) orrejected as foreign (histoincompatible). For instance, within the classH region, three loci, i.e., HLA-DR, DQ and DP are known to expressfunctional products. Pairs of A and B genes within these three lociencode heterodimeric protein products which are multi-allelic andallorcactive. In addition, combinations of epitopes on DR and/or DQmolecules are recognized by alloreactive T cells. This reactivity hasbeen used to define “Dw” types by cellular assays based upon the mixedlymphocyte reaction (MLR). It is contemplated that matching of the HLAtype of the reference biological sample with the mammary fluid samplemay be used to determine whether the mammary fluid sample originatedfrom the donor.

One nucleic acid typing method for the identification of these alleleshas been restriction fragment length polymorphism (RFLP) analysisdiscussed herein (see, also, U.S. Pat. No. 6,670,124).

In addition to restriction fragment length polymorphism (RFLP), anotherapproach is the hybridization of PCR amplified products withsequence-specific oligonucleotide probes (PCR-SSO) to distinguishbetween HLA alleles (see, Tiercy et al., (1990) Blood Review 4:9-15).This method requires a PCR product of the HLA locus of interest beproduced and then dotted onto nitrocellulose membranes or strips. Theneach membrane is hybridized with a sequence specific probe, washed, andthen analyzed by exposure to x-ray film or by colorimetric assaydepending on the method of detection. Similarly to the PCR-SSPmethodology, probes are made to the allelic polymorphic area responsiblefor the different HLA alleles. Each sample must be hybridized and probedat least 100-200 different times for a complete Class I and II typing.Hybridization and detection methods for PCR-SSO typing include the useof nonradioactive labeled probes, microplate formats, and the like (see,e.g., Saiki et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86: 6230-6234;Erlich et al. (1991) Eur. J. Immunogenet. 18(1-2): 33-55; Kawasaki etal. (1993) Methods Enzymol. 218:369-381), and automated large scale HLAclass II typing (see, e.g., U.S. Pat. No. 6,670,124).

Another typing method comprises sequence specific primer amplification(PCR-SSP) which may be used in the methods of the invention (see, Olempand Zetterquist (1992) Tissue Antigens 39: 225-235). In PCR-SSP, allelicsequence specific primers amplify only the complementary templateallele, allowing genetic variability to be detected with a high degreeof resolution. This method allow determination of HLA type simply bywhether or not amplification products (collectively called an“amplicon”) are present or absent following PCR. In PCR-SSP, detectionof the amplification products is usually done by agarose gelelectrophoresis followed by ethidium bromide (EtBr) staining of the gel(see, e.g., U.S. Pat. No. 6,670,124).

Another HLA typing method is SSCP—Single-Stranded ConformationalPolymorphism. Briefly, single stranded PCR products of the different HLAloci are run on non-denaturing Polyacrylamide Gel Electrophoresis(PAGE). The single strands will migrate to a unique location based ontheir base pair composition. By comparison with known standards, atyping can be deduced. It is the only method that can determine truehomozygosity, (see, e.g., U.S. Pat. No. 6,670,124) (Orita et al., Proc.Nat. Acad. Sci 86:2766-2770 (1989)).

The identification of a DNA sequence can be made without anamplification step, based on polymorphisms including restrictionfragment length polymorphisms (“RFLP”) in a subject. Hybridizationprobes are generally oligonucleotides which bind through complementarybase pairing to all or part of a target nucleic acid. Probes typicallybind target sequences lacking complete complementarity with the probesequence depending on the stringency of the hybridization conditions.The probes are typically labeled directly or indirectly, such that byassaying for the presence or absence of the probe, one can detect thepresence or absence of the target sequence. Direct labeling methodsinclude radioisotope labeling, such as with 32P or 35S. Indirectlabeling methods include fluorescent tags, biotin complexes which may bebound to avidin or streptavidin, or peptide or protein tags. Visualdetection methods include photoluminescents, Texas red, rhodamine andits derivatives, red leuco dye and 3,3′,5,5′-tetra-methylbenzidine(TMB), fluorescein, and its derivatives, dansyl, umbelliferone and thelike or with horse radish peroxidase, alkaline phosphatase and the like(see, e.g., U.S. Patent Publication No. 20040253594, U.S. PatentPublication No. 20050123947, which are incorporated by reference).

One or more additional restriction enzymes and/or probes and/or primerscan be used. Additional enzymes, constructed probes, and primers can bedetermined by routine experimentation by those of ordinary skill in theart and are intended to be within the scope of the invention.

Although the methods described herein may be in terms of the use of asingle restriction enzyme and a single set of primers, the methods arenot so limited. One or more additional restriction enzymes and/or probesand/or primers can be used, if desired. Additional enzymes, constructedprobes and primers can be determined through routine experimentation,combined with the teachings provided and incorporated herein.

The reagents suitable for applying the methods of the invention may bepackaged into convenient kits. The kits provide the necessary materials,packaged into suitable containers. Typically, the reagent is a PCR set(a set of primers, DNA polymerase and 4 nucleoside triphosphates) thathybridize with the gene or loci thereof. Typically, the PCR set isincluded in the kit. Typically, the kit further comprises additionalmeans, such as reagents, for detecting or measuring the detectableentity or providing a control. Other reagents used for hybridization,prehybridization, DNA extraction, visualization etc. may also beincluded, if desired.

Other Identity Markers Profiles

It is further contemplated that the mammary fluid sample may be testedfor self-antigens (or other peptides and polypeptides) present in themammary fluid to establish a self-antigen profile (identity markerprofile). The self-antigen profile of the mammary fluid sample will becompared to the reference self-antigen profile for the individual human.A match or identity of the self-antigen profile will indicate that themammary fluid was obtained from the specific subject.

The various antigens that determine self are encoded by more than 40different loci, such as the major histocompatibility complex (MHC), alsocalled the human leukocyte antigen (HLA) locus, and the blood groupantigens, such as ABO.

Methods are known in the art for screening humans for ABO blood grouptype. The blood-group antigens are expressed on red blood cells,epithelial cells and endothelial cells.

Testing for HLA type can be conducted by methods known in the art suchas serological and cellular typing.

It is contemplated that the antigens could be identified by amicrocytotoxicity test. In this test, white blood cells are distributedin a microtiter plate and monoclonal antibodies specific for class I andclass II MHC antigens are added to different wells. Thereafter,complement is added to the wells and cytotoxicity is assessed by uptakeor exclusion to various dyes (e.g., trypan blue or eosinY) by the cells.If the white blood cells express the MHC antigen for a particularmonoclonal antibody, then the cells will be lysed on addition ofcomplement and these dead cells will take up the dye (see, Terasaki andMcClelland, (1964) Nature, 204:998 and U.S. Pat. No. 6,670,124). HLAtyping based on antibody-mediated microcytotoxicity can thus indicatethe presence or absence of various MHC alleles (See Kuby Immunology 4thEd., Freeman and Company, pp 520-522).

The detection of antigens may be selected from, but is not limited to,enzyme-linked immunosorbent assay, solid phase radiobinding immunoassayswhere the antibodies may be directed against soluble antigens or cellsurface antigens, autoradiography, competitive binding radioimmunoassay,immunoradiometric assay (IRMA) electron microscopy, peroxidaseantiperoxidase (PAP) labeling, fluorescent microscopy, alkalinephosphatase labeling and peroxidase labeling.

In the case where the detection method (s) use optical microscopy, thecells from the biological sample or the mammary fluid sample are mountedand fixed on a microscope slide. In this case, the step of detecting thelabelled antibody is detecting a resulting colouration of theself-antigen with an optical microscope (see, e.g., U.S. Pat. No.6,376,201).

The following example provides an embodiment of the methods describedherein and should not be understood as restrictive.

Example 1 Testing of a Human Breast Milk Donor

A woman who wishes to donate her breast milk will provide a biologicalreference sample prior to (or at the time of) her first donation. Thebiological sample will include a convenient tissue type, e.g., blood,cheek cell, hair etc. The sample will be donated under supervision ofanother individual(s), e.g., bank milk personnel. The sample will belabeled for later reference. The reference sample will be tested for aspecific marker profile, e.g., nucleic acid and/or peptide profile. Thesample will be tested for one or more markers. Results of the tests willbe stored, e.g., on a computer-readable medium for future reference. Anyremaining sample will be stored. The woman can also be screened (usingthe reference sample or another sample) for, e.g., drug use, viruses,bacteria, parasites, and fungi etc., to determine her health. The womanwill be given a label corresponding to the reference sample to keep withher and use with her donated milk.

Alternatively, the sample will be stored without testing, and will betested at a later date, for example, together with the donated breastmilk.

The woman will express her milk for donation and either forward the milkto the milk bank or processing facility or store the milk in herrefrigerator, e.g., the freezer, for donation with other samples at alater date. The donated milk will be labeled with the label given to thewoman and matching the reference sample.

A sample of the donated milk that arrives at the milk bank or processingfacility will be tested for at least one of the same markers as thereference sample. The marker profile of the reference sample will becompared to the marker profile of the donated milk sample. If theprofiles will match, the identity of the donor will be confirmed. If theprofiles will not match, the results will be an indication that thedonated milk is contaminated with another woman's milk or that it doesnot come from the woman whose reference sample was taken.

The milk whose provenance (i.e., origin) will be confirmed by thematched profiles will be further processed, e.g., pasteurized, e.g.,into human milk fortifiers, standardized human milk compositions, and/orhuman lipid compositions. Such compositions will be administered tohuman infants, e.g., premature infants, whose mothers may not be able toprovide them with adequate nutrition.

The reference sample and/or results of the reference sample tests willbe stored for any future donation by the corresponding mother.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for determining whether a donatedmammary fluid was obtained from a specific subject, the methodcomprising: (a) testing a donated biological sample from the specificsubject to obtain at least one reference identity marker profile for atleast one marker; (b) testing a sample of the donated mammary fluid toobtain at least one identity marker profile for the at least one markerin step (a); (c) comparing the identity marker profiles, wherein a matchbetween the identity marker profiles indicates that the mammary fluidwas obtained from the specific subject; and (d) processing the donatedmammary fluid whose identity marker profile has been matched with areference identity marker profile, wherein the processed donated mammaryfluid comprises a human protein constituent of 11-20 mg/mL; a human fatconstituent of 35-55 mg/mL; and a human carbohydrate constituent of70-120 mg/mL.
 2. The method of claim 1, wherein the mammary fluid ishuman breast milk.
 3. The method of claim 1 wherein the processingcomprises: (a) filtering the milk; (b) heat-treating the milk; (c)separating the milk into cream and skim; (d) adding a portion of thecream to the skim; and (e) pasteurizing.
 4. The method of claim 1,wherein the composition further comprises one or more constituentsselected from the group consisting of: calcium, chloride, copper, iron,magnesium, manganese, phosphorus, potassium, sodium, and zinc.
 5. Themethod of claim 1, wherein processing comprises separating the milk intoa cream portion and a skim portion, processing the cream portion, andpasteurizing the cream portion.
 6. The method of claim 1, furthercomprising nucleic acid typing, wherein the nucleic acid typingcomprises a method selected from the group consisting of: STR analysis,HLA analysis, multiple gene analysis, and a combination thereof.
 7. Themethod of claim 1, wherein the donated mammary fluid is frozen.
 8. Themethod of claim 1, wherein the mammary fluid sample comprises a mixtureof one or more mammary fluid samples.
 9. The method of claim 1, whereinthe donated biological sample is selected from a group consisting of:milk, saliva, buccal cell, hair root, and blood.
 10. The method of claim1, wherein steps (a) through (c) are carried out at a human breast milkdonation center or at a milk processing facility.
 11. The method ofclaim 1, wherein steps (a) and (b) are carried out at differentfacilities.
 12. The method of claim 11, wherein step (a) is carried outat a human breast milk donation facility and step (b) is carried out ata milk processing facility.
 13. A method for processing a donated humanbreast milk obtained from a specific subject comprising: (a) testing adonated biological sample from the specific subject to obtain at leastone reference identity marker profile for at least one marker; (b)testing a sample of the donated human breast milk to obtain at least oneidentity marker profile for the at least one marker in step (a); (c)comparing the identity marker profiles of steps (a) and (b), wherein amatch between the identity marker profiles indicates that the donatedhuman breast milk was obtained from the specific subject; (d) processingthe donated human breast milk whose identity marker profile has beenmatched with a reference identity marker profile, wherein the processingcomprises: (i) filtering the donated human breast milk; (ii) heattreating the donated human breast milk; (iii) separating the donatedhuman breast milk into cream and skim; (iv) adding a portion of thecream to the skim to form a human milk composition; and (v) pasteurizingthe human milk composition to produce a processed human breast milkcomposition; and wherein the processed donated human breast milkcomprises a human protein constituent of 11-20 mg/mL; a human fatconstituent of 35-55 mg/mL; and a human carbohydrate constituent of70-120 mg/mL.
 14. A processed human milk composition suitable foradministration to an infant made by the process of claim
 13. 15. Themethod of claim 13, wherein the method further comprises adding to theprocessed human breast milk one or more constituents selected from thegroup consisting of: calcium, chloride, copper, iron, magnesium,manganese, phosphorus, potassium, sodium, and zinc.
 16. The method ofclaim 13, wherein the testing of the donated human breast milk of step(b) and the testing of the donated biological sample of step (a)comprises nucleic acid typing selected from the group consisting of: STRanalysis, HLA analysis, multiple gene analysis, and a combinationthereof.
 17. The method of claim 13, wherein the donated biologicalsample is selected from a group consisting of: milk, saliva, buccalcell, hair root, and blood.
 18. The method of claim 13, wherein steps(a) through (c) are carried out at a human breast milk donation centeror at a milk processing facility.
 19. The method of claim 13, whereinsteps (a) and (b) are carried out at different facilities.
 20. Themethod of claim 19, wherein step (a) is carried out at a human breastmilk donation facility and step (b) is carried out at a milk processingfacility.